The invention relates to compositions of alpha1-proteinase inhibitor (A1PI) and methods of making and use.
Mature alpha1-proteinase inhibitor (A1PI) is a single chain glycoprotein composed of 349 amino acids and 12% carbohydrate (weight %) (see, e.g., Coan et al., Vox Sanguinis, 48:333-342 (1985)). Heterogeneity of A1PI is due to posttranslational modifications and covalent linkage of 3 complex N-glycans to asparagines 46, 83 and 2471,2. The high negative charge of A1PI is the result of multiple sialic acid residues on N-glycans leading to multiple isoforms of A1PI (M1 to M8) that become visible after electrophoresis3-8. Two minor cathodal isoforms, M7 and M8, are the result of N-terminal truncation of 5 amino acids including negatively charged glutamic and aspartic acids9.
A1PI belongs to the family of serpins that inhibit serine proteases. Neutrophil elastase, an enzyme which degrades a number of proteins of the interstitial extracellular matrix, is a serine protease that is inhibited by A1PI. In patients with inherited A1PI deficiency the balance between neutrophil elastase and A1PI is disturbed, which increases their risk of developing lung emphysema. In these patients, elastase released from neutrophils in the lower respiratory tract escapes neutralization by A1PI with consequent chronic destruction of lung parenchyma, which becomes clinically apparent in the third to fourth decade of life10.
To slow down the progression of emphysema in patients with A1PI deficiency the protease-anti-protease balance is restored by life-long augmentation therapy with highly purified plasma-derived A1PI concentrates which raise A1PI in the circulation11. Three different products (PROLASTIN® (Alpha1-Proteinase Inhibitor (Human)), ARALAST® (Alpha1-Proteinase Inhibitor (Human)), and ZEMAIRA® (Alpha1-Proteinase Inhibitor (Human))) are approved by the US FDA for the treatment of A1PI deficiency. These products are manufactured from large pools of ˜10,000 liters of human plasma12-14. Upstream manufacturing and downstream purification processes including pathogen-reduction steps vary to differing extents between products12,15,16. After removal of the immune-globulin containing plasma fractions various sequential steps of the Cohn/ethanol fractionation, including chromatography, protein precipitation and co-precipitation followed by resolubilization, diafiltration for buffer exchange, concentration steps and viral reduction steps, take advantage of the physicochemical properties of A1PI to concentrate A1PI into an intermediate fraction. This fraction is used for subsequent downstream purification. A1PI is therefore exposed to different physicochemical conditions and to a variety of enzymes during the manufacturing process.
Available A1PI concentrates have a purity of >80% and specific activities ranging from 0.6 to 1.0 U A1PI/mg protein with different plasma protein impurity profiles. High resolution isoelectric focusing (IEF) analysis of A1PI present in A1PI products has revealed differences in the IEF band pattern of glycolsoforms and raised questions from patients, physicians and the FDA. This difference in electrophoretic mobility was not caused by differences in N-glycan profiles, but mainly by varying degrees of C-terminal lysine truncation at position 394 from the A1PI molecule adding an additional negative charge to the protein8,17,18. The percent of A1PI C-terminal truncation as compared to total A1PI protein differed in the three approved products. ARALAST® (Alpha1-Proteinase Inhibitor (Human)) showed approximately 60% (67%) truncated A1PI, while PROLASTIN® (Alpha1-Proteinase Inhibitor (Human)) showed 2% truncated A1PI and ZEMAIRA® (Alpha1-Proteinase Inhibitor (Human)) showed 6% truncated A1PI.
Basic carboxypeptidases are a group of enzymes that specifically cleave C-terminal basic amino acids (arginine or lysine) from peptides and proteins leading to an increased negative charge of the protein19. Basic carboxypeptidases are involved in a variety of biological processes such as food digestion, inactivation of complement components20, inhibition of fibrinolysis21 and processing of peptide hormones22.
In this application, we show that A1PI belongs to the group of proteins that are a substrate for basic carboxypeptidases and that the C-terminal truncated form of A1PI also occurs naturally. We also describe the manufacturing conditions which result in the removal of the positively charged C-terminal lysine of A1PI by carboxypeptidases.